Mitogen activated protein (MAP) kinase pathways (a cascade of three successive protein kinases) are a common molecular signal transduction system used to mediate eukaryotic cell responses to their environment. MAP Kinase pathways are used by mammalian cells to mediate cell growth, differentiation, apoptosis, stress response and immune response. They are also used in plants for environmental sensing. In different cells, this core pathway is reprogrammed to yield a tailored response.
Our invention provides a method to systematically reprogram MAP kinase signaling responses. The invention allows one to engineer and modify MAP kinase signaling behavior such that it is optimized for new target behaviors. This method can be used, for example, to engineer into yeast modified control systems for regulating metabolic processes, such as in engineered biofuel production.
Our method uses engineered feedback loops to modify pathway response. Much of the diversity in natural MAP kinase response behaviors is due to the fact that individual examples have distinct feedback loops overlaid on the core linear kinase cascade. As in electronic circuits, these feedback loops profoundly alter behavior—specific feedback architectures can lead to distinct classes of responses. By building specific synthetic feedback loops, we show that we can systematically engineer MAP kinase pathway behavior in living cells.
Our method for building novel feedback loops relies on the fact that MAP kinase pathways are organized by scaffold proteins—proteins (or a complex) that bind to the pathway members and facilitate their communication with one another and prevent miscommunication with incorrect molecules. Thus the scaffold is a hub that organizes the wiring of the pathway. In our method, by conditionally recruiting new pathway modulators to the scaffold, we can build positive or negative feedback loops (FIG. 2a).
In an illustrative embodiment, the method comprises three general classes of parts: 1) pathway modulators, 2) recruitment interaction pair, and 3) pathway induced promoter. The general logic is that one constructs a gene that consists of a pathway modulator that is fused to one of the interaction pair partners, and places this under the control of a promoter that is only activated when the MAPK pathway is turned activated. One then fuses the other interaction pair partner domain to the scaffold protein. Thus when the pathway is activated, it will lead to expression of the modulator-interaction domain fusion protein, which will then be recruited to the scaffold. When recruited to the scaffold, the modulator can strongly exert its effect; if this was a positive modulator, it would generate a positive feedback loop; if it was a negative modulator, it would generate a negative feedback loop.
In proof of principle experiments, we have used a variety of modulators and interaction pairs to build such illustrative feedback loops in the yeast mating MAP kinase pathway, including the positive activator of MAPKKK, Ste50, the negative modulators Msg5 (MAPK phosphatase—from yeast), OspF (MAPK phosphothreonine lyase—from Shigella), and YopJ (MAPK acetylase—from Yersinia), and the PDZ-peptide and pairs leucine-zipper heterodimerization interaction pairs; of course, alternative modulators and interaction pairs maybe substituted.
As detailed further below, our scaffold-based engineering method can be practiced in numerous variations. For example, multiple feedback loops can be layered to achieve more complex behaviors. Also, decoy interaction domains can be introduced to compete with the recruitment of pathway modulators to the scaffold. Together, these simple variations allow for more generation of more complex behaviors.
We have used these methods to alter MAP kinase pathways to show several distinct and useful classes of cellular behavior, including threshold sensing (ultrasensitve response), adaptation to continued stimulation, pulse response, delayed response, and accelerated response. In several cases we have demonstrated that by varying parameters such as recruitment interaction affinity or promoter strength, we can systematically tune the precise response behavior.
Our methods can be applied to systematically tune the behavior of any cell containing a MAP kinase pathway that is organized by a scaffold protein, or other comparable protein assemblies. Using the basic design framework outlined herein, and by exploring a finite range of system parameters, the pathway response behavior can be tuned as desired. Our methods are also suitable for constructing small combinatorial variants.
The methods can be used in any cell that uses MAPK signaling for a process of interest. Often the endogenous MAPK pathway behavior in a cell, while in principle useful for a desired function, might have quantitative response behaviors that are incompatible with the target function. Our methods can be used to tune the behavior such that it is optimized. This method could be useful for a wide range of applications, including: a) engineering cell lines with specifically tuned MAP kinase response behaviors optimized for drug screening; b) engineering therapeutic immune cells that detect tunable levels of antigen and respond in a tunable manner; c) engineering stem cells that respond to stimuli with precisely tunable differentiation or apoptotic responses; d) engineering plant cells that can detect specific signals and respond in tunable way, for example, detection of pathogens or noxious chemicals, or for production or agricultural purposes that require input controlled induction; and e) engineering yeast cells to provided control systems for regulating metabolic processes, such as in engineered biofuel production. In short, our invention provides general methods for achieving tunable control over a protein signaling pathway, by using recruitment pairs to introduce novel regulatory feedback loops into MAPK signaling pathways.